1. Field of the Invention (Technical Field)
The present invention relates to three-dimensional light traps for reflective particles.
2. Background Art
The last few decades have brought about a revolution in our understanding of physical processes at the microscopic level in virtually every scientific discipline. The impact of this upheaval has perhaps been greatest in the area of molecular biology. For example, even our perception of how life itself is constituted at the physical level has changed radically with the discovery and then characterization of DNA. Research into biological systems has been motivated at least in part by a desire to better understand how to treat and cure disease and extend human life. These research advances have been made possible by a concurrent revolution in biological instrumentation. As in any scientific field, there has been a synergism between instrumentation and research, with new analytical tools opening up new possibilities for research, and new scientific discoveries and theories driving the demand for more powerful, more sensitive, and novel scientific instrumentation.
Research at the microscopic level in biological systems has been hampered by the fact that it is often difficult in practice to isolate a biological particle of interest from the laboratory environment or contaminants. Significant progress is being made in this area, however, thanks to the advent of a technique known as "optical trapping." This technique uses light particles, or photons, to hold or "trap" small particles of transparent or semi-transparent matter.
Optical trapping is based on the principle of conservation of momentum and is illustrated in FIG. 1(a), which illustrates the case of a small, spherical transparent particle in the presence of nonuniform photon flux, such as the gaussian distribution of a laser beam. For a transparent particle, the fraction of light which is scattered is typically small, and most of the light will be refracted through the particle instead. If the index of refraction of the particle is greater than that of the surrounding medium, then the light rays will be refracted towards the normal of the surface as they enter the particle, and away from the normal as they exit it, in accordance with standard geometrical optical theory. The light has undergone a net change in direction, and thus there has been a net change in the photons' momentum. This is illustrated for photons entering the right hand side of the particle by the vector inset in FIG. 1(a), where the initial and final momenta are designated by the subscripts i and f, respectively. Since momentum must always be conserved, the resulting change in a photon's momentum must be compensated for by an equal and opposite change in the momentum of the particle itself. For the vector inset in FIG. 1a, this corresponds to a net change in the momentum of the particle to the right, indicated by the vector labeled "reaction force." Of course, light rays entering the left hand side of the particle have the opposite effect, i.e., they tend to push the particle to the left. If the photon flux were homogeneous, then these effects would cancel each other out completely, and the particle would not experience any net push to the right or left. In the case of a light gradient assumed here, however, there is a net change in the particle's momentum towards the center of the light beam. Clearly, a stronger field will produce a proportionally greater trapping effect.
In addition to the two dimensional (or lateral) trapping force discussed above, there is an additional force which is longitudinal in orientation. FIG. 1(b) shows how the direction of light rays changes when a refracting particle is situated near the beam focus. A straightforward momentum conservation (vector) analysis analogous to the one done in connection with FIG. 1(a) shows that the reaction force acting upon the particle in this case is once again directed towards the focal point. Thus, the lateral trapping force and the longitudinal force act in concert to push the particle towards the center of the light beam where it eventually comes to equilibrium.
To reiterate, optical trapping of transmissive particles is based on the principle that light imparts a change in momentum when it is refracted through a small particle. This change in momentum imparts a small force on the particle. If the light is uniform, then the refraction from the particle is the same in all directions, and no net force is imparted. However, if there is a strong intensity gradient in the light (usually laser) beam, then the forces can be unbalanced if the particle is not centered in the optical beam. While the net force is relatively small, for microscopic particles the mass of the particle is low enough that the net force is sufficient to lock it in place.
Optical trapping was first demonstrated by Ashkin at Bell Labs in the late 1960's, A. Ashkin, "Acceleration and trapping of particles by radiation pressure", Phys. Rev. Lett. 24:156 (1970), but not applied to biological systems until relatively recently, A. Askin, et al., "Optical trapping and manipulation of viruses and bacteria", Science 235:1517 (1987); A. Ashkin, et al., "Optical trapping and manipulation of single cells using infrared laser beams", Nature 330:769 (1987); and U.S. Pat. No. 4,893,886, to A. Ashkin, et al., entitled "Non-destructive optical trap for biological particles and method of doing same", issued Jan. 16, 1990. This art has been studied and practically applied in a variety of ways. T. C. B. Schut, et al., "Experimental and theoretical investigation on the validity of the geometrical optics model for calculating the stability of optical traps", Cytometry 12:479 (1991); G. Roosen, et al., "The TEM.sub.01 * mode laser beam--a powerful tool for optical levitation of various types of spheres", Opt. Comm. 26:432 (1978); and Cell Robotics, Inc., LaserTweezers.TM. device.
Although biological particles are generally not spherical, the same physical principles governing optical trapping apply to them. An infrared laser is generally used as the trapping laser, since biological materials typically do not absorb in the IR, thus minimizing the chance that the biological samples might be inadvertently damaged or destroyed. Instrumentation based on the principle of optical trapping is commercially available from Cell Robotics, Inc., and is sold under the trademark LaserTweezers. A schematic of this product is shown in FIG. 6. The device consists essentially of a computer-controlled, motorized XY stage, a Z-drive, a laser module and a camera, all of which are directly mounted onto a microscope. The laser light is steered through the microscope so that the beam fills the rear aperture of the objective, resulting in a tightly focused beam suitable for optical trapping. The trap is formed at the focal point of the laser beam, as discussed above. Since the laser alignment is fixed, moving the trapped particle within the XY plane is accomplished by moving the XY stage. The stage has a resolution of 0.1 micron and a repeatability of 1 micron, so that measurements can be controlled. Motion along the Z-axis, on the other hand, is controlled with the Z-drive which moves the microscope objective up and down. The contents of the manipulation chamber can be viewed with an eyepiece or a camera, both of which are mounted to the microscope and are protected by an infrared blocking filter.
Although the LaserTweezers optical trapping technique is a very useful one, its utility is generally restricted to those situations in which the object to be trapped is at least semi-transparent and has an index of refraction greater than that of the surrounding medium. This is because for a reflective particle, the forces act in exactly the opposite direction. Instead of being trapped, the reflective particle is pushed away. There are limited exceptions to this, however. Roosen, et al. have used a TEM.sub.01 * mode laser beam to optically levitate metallic spheres. This technique, however, can only be used provided that the laser beam diameters are in certain mathematical proportions. In addition, two laser beams may be required in some situations for optical levitation to occur. Also, Svoboda and Block have demonstrated that small metallic particles can be trapped with optical tweezers, but only when the particles have radii much smaller than that of the wavelength of the trapping light (the so-called Rayleigh regime). K. Svoboda, et al., "Optical trapping of metallic Rayleigh particles", Optics Lett. 19:930 (1994). For example, stable traps were formed with gold and latex particles having diameters of 36 and 38 nm, respectively.
Thus, the most common optical trapping techniques rely on the particle being transmissive to the light. However, for a reflective particle, the forces operate in exactly the opposite direction, and instead of the light beam trapping the particle, it is accelerated away rapidly. Only very small particles (those that are smaller than the wavelength of light) can be trapped using a single light beam. K. Svoboda, et al., "Optical trapping of metallic Rayleigh particles", Optics Lett. 19:930 (1994). Roosen, et al. have used TEM.sub.01 * laser beams to create small traps for reflective particles. However, these beams are determined by the mode pattern of the laser, and are not easily matched to the particle size in any convenient fashion.
To date, only one technique has been proposed which addresses the problem of how to optically trap reflecting particles or particles which have an index of refraction less than that of surrounding medium. K. Sasaki, et al., "Optical trapping of a metal particle and a water droplet by a scanning laser beam", Appl. Phys. Lett. 60:807 (1992); and U.S. Pat. No. 5,212,382, to K. Sasaki, et al., entitled "Laser trapping and method for applications thereof", issued May 18, 1993. Sasaki, et al. have disclosed the method of FIG. 1(c), which involves scanning a focused laser beam around the particle to be trapped. The scanned beam forms a "reflective cage of light" around the particle, effectively confining it within the light cage. The case of reflecting particles is analogous to the solar wind phenomenon where photons act to push away particles. Likewise, transmissive particles with indices of refraction lower than that of the surrounding medium are trapped as well, as can be seen by a conservation of momentum analysis analogous to that presented in connection with FIG. 1(b). In this case, the momentum imparted to the particle pushes it away from regions of higher light intensity, or in other words, towards the center of the "doughnut hole" defined by the scanning laser beam.
The method of Sasaki, et al. suffers from limitations, however. The laser must be scanned fast enough to overcome diffusion of the particle out of the light cage. Thus, the viscosity of the solvent and the size of the particles determine which combinations of particles and solvent media can be used. There is the cost and complexity introduced by the scanner and associated hardware. In addition to the elements needed to inject the laser beam into the microscope, a scanning mirror must be included in the optical system. This mirror must operate at a high enough bandwidth that the particle cannot escape in the time it takes to complete a circle. Further, the scan system can introduce vibrations or other errors into the system.
The present invention circumvents the restrictions of the prior art light cage apparatuses to permit direct and straightforward manipulation of reflective particles of many sizes without a complex scanning system.